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Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity

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Abstract

Mycoparasitic Trichoderma strains secrete a complex set of hydrolytic enzymes under conditions related to antagonism. Several proteins with proteolytic activity were detected in culture filtrates from T. harzianum CECT 2413 grown in fungal cell walls or chitin and the protein responsible for the main activity (PRA1) was purified to homogeneity. The enzyme was monomeric, its estimated molecular mass was 28 kDa (SDS-PAGE), and its isoelectric point 4.7–4.9. The substrate specificity and inhibition profile of the enzyme correspond to a serine-protease with trypsin activity. Synthetic oligonucleotide primers based on N-terminal and internal sequences of the protein were designed to clone a full cDNA corresponding to PRA1. The protein sequence showed <43% identity to mammal trypsins and 47–57% to other fungal trypsin-like proteins described thus far. Northern analysis indicated that PRA1 is induced by conditions simulating antagonism, is subject to nitrogen and carbon derepression, and is affected by pH in the culture media. The number of hatched eggs of the root-knot nematode Meloidogyne incognita was significantly reduced after incubation with pure PRA1 preparations. This nematicidal effect was improved using fungal culture filtrates, suggesting that PRA1 has additive or synergistic effects with other proteins produced during the antagonistic activity of T. harzianum CECT 2413. A role for PRA1 in the protection of plants against pests and pathogens provided by T. harzianum CECT 2413 is proposed.

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Authors and Affiliations

  1. 1IBVF-CIC Isla de la Cartuja, CSIC/Universidad de Sevilla, Sevilla, Spain

    Belen Suarez & Antonio Llobell

  2. Newbiotechnic S.A, Sevilla, Spain

    Manuel Rey

  3. Instituto de Agricultura Sostenible, CSIC, Cordoba, Spain

    Pablo Castillo

  4. Centro Hispano-Luso de Investigaciones Agrarias, Universidad de Salamanca, Salamanca, Spain

    Enrique Monte

  5. Centro de Investigaciones Científicas Isla de la Cartuja, Instituto de Bioquímica Vegetal y Fotosíntesis, Avda. Américo Vespucio s/n, Isla de la Cartuja, 41092, Sevilla, Spain

    Antonio Llobell

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  1. Belen Suarez
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  2. Manuel Rey
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  3. Pablo Castillo
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  4. Enrique Monte
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  5. Antonio Llobell
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Correspondence to Antonio Llobell.

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Suarez, B., Rey, M., Castillo, P. et al. Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity. Appl Microbiol Biotechnol 65, 46–55 (2004). https://doi.org/10.1007/s00253-004-1610-x

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